Survival of patients with malignant glioma remains poor despite the availability of surgical debulking, radiation therapy, and chemotherapeutic regimens. Progress in applying gene therapy to the treatment of cancer provides an additional strategy which may prove effective in combination with more standard therapies. NUREL-C2 is a completely inactivated herpes simplex virus (HSV)- based gene transfer vehicle that expresses the four novel therapeutic proteins ICP0, thymidine kinase, connexin-43 and TNFalpha which work in concert to kill tumor cells when used in combination with intravenous administration of the anti-cancer drug ganciclovir (GCV) and radiosurgery. Animal experiments using this combination of gene and conventional therapies to treat intracerebral implants of radiosensitive human glioblastoma cells have resulted in excellent tumor control and improved survival. To establish the maximum the maximum potential of this approach, additional preclinical studies are proposed to optimize the contributions of each component to the combined treatment and to evaluate efficacy in models of radioresistant human glioblastoma (Aim 1). The vector and combined therapy will be systematically tested for safety and dose-limiting toxicity in normal mice and rhesus monkeys to expand our current results (Aim 2). A Phase I clinical trial is proposed with two consecutive components involving a) pre- and post-surgical intracranial NUREL-C2 inoculation followed by GCT treatment, and b) stereotactic NUREL-C2 delivery into the tumor with maintenance on GCV and gamma knife radiosurgery two days later. Using a battery of molecular, serological, imaging and clinical tests, patients will be evaluated for adverse effects of viral vector implantation, vector toxicity prior to, during, and after GCV treatment, short-term vector distribution and transgene expression in the tumor, metabolic activity of the tumor, and imaging responses to therapy. Safe vector dose will be determined in the first aim of the trial by dose escalation between consecutive groups of 3 patients. In the second arm, potential changes in toxicity profile and safe dose due to the combination with radiosurgery will be identified. Concurrent manifestations of efficacy will be recorded (Aim 3). In the final Aim, the therapeutic potential of HSV vectors expressing radiosensitizing genes or novel genes from Projects 1 and 2 will be tested for effectiveness in glioma models. Effective genes will be incorporated into NUREL-C2 and the new derivatives tested for improved cytocidal qualities in vitro and efficacy in vivo to arrive at an optimally effective gene transfer agent for the treatment of malignant glioma (Aim 4).